1. Field of the Invention
This invention relates to dual-wavelength absorbance detection in chemical analysis and more particularly to dual-wavelength optical absorption detectors used in continuous flow analysis, flow injection analysis, colorimetry and liquid and gas chromatography.
2. Description of the Related Art
Developments in the techniques and methodologies of chemical analysis have advanced rapidly over the last several years, especially for laboratory instruments; however, the evolution of state-of-the-art hardware designed specifically for the measurement of absorption in continuous flow analysis, FIA (flow injection analysis), colorimetry or HPLC (high performance liquid chromatography) has lagged far behind. Currently, dual-wavelength absorption analysis is done with complex instruments that are not specifically designed for dual-wavelength absorption detection such as an expensive, bulky, bench-top spectrophotometer. Or, it is done with single beam, dual-wavelength absorption detectors which have inherent design limitations that increase the cost of sampling and/or reduce the performance of the detector.
In this specification, dual-wavelength absorption detection may be considered to fall within the field of optical detection; however, the methods and apparatus discussed herein may also include absorption detection in the ultraviolet and infrared region as well as the visible region of the spectrum. Dual-wavelength absorption detection is based upon the Lambert-Beer law which states that the amount of light absorbed by a given substance in solution (Absorbance) is proportional to the intensity of incident light and to the concentration of the absorbing species. This is expressed mathematically as: EQU A=abc=log I.sub.0 /I=log 1/T (1)
where:
A=absorbance, PA1 a=molar absorptivity in liters per mole-centimeter, PA1 b=light path length in centimeters, PA1 c=concentration of substance in moles per liter, PA1 I.sub.0 =intensity of radiation detected with a nonabsorbing sample placed along a light path of length "b" at a certain wavelength, PA1 I=intensity of radiation detected with an absorbing sample having a concentration "c" placed along the light path of length "b" at the same wavelength, and PA1 T=I/I.sub.0 =transmittance.
It may also be expressed as a log ratio between a non-absorbing species and an absorbing species where:
The Lambert-Beer law requires the use of a single beam, single-wavelength detector to provide a measurement of "absorbance" for the absorbing species when a nonabsorbing species is used to establish the reference level of illumination. One widely used variation from the Lambert-Beer law may be used in analytical chemistry to provide an approximate indication of the absorbance of a species at a specific wavelength without using a second nonabsorbing species to establish the reference level of illumination. The resulting measurement may not be an exact measurement of absorbance as defined by formula (1) because this variation does not rigorously follow the Lambert-Beer Law; the measurement using this variation is referred to in analytical chemistry as an "absorption" measurement. This widely used variation is called single beam, dual-wavelength, absorption detection. In this variation, a single sample or species is irradiated with a beam having at least two different wavelengths, a measuring wavelength and a reference wavelength, preferably the source supplies the wavelengths to have equal intensity. The measuring wavelength is selected in the region of the spectrum where the species to be monitored absorbs, whereas the reference wavelength is selected where the species has little or no absorbance. The measuring wavelength would be approximately equivalent to the "I" of formula (1), the reference wavelength would be approximately equivalent to "I.sub.0 " of the formula, and the log of the ratio I.sub.0 /I is in units of "absorption". Preferably, the two wavelengths would be selected to be near each other in the spectrum.
In some dual-wavelength absorption measurements, the reference wavelength may have some absorbance in the species but to a lesser extent than the measuring wavelength. In this case, the absorption measurement would reflect the log ratio of the difference in the absorption of optical energy by the species at both wavelengths. Thus, with a known chemistry, the detected "absorption" value of the species, i.e., the value or amplitude of the output signal using a particular detector, would be a known amount. If there is a deviation from this known absorption value, then the chemistry of the species would have changed.
The single beam, dual-wavelength, absorption detector is widely used for performing chemical analysis involving the sampling of a consecutive flow of materials passing through the detector. FIG. 1 depicts the output of such a detector over time. Absorption peaks 1 through 8 represent the absorption values of various species which are separated in the continuous-flow sample stream by a volume of carrier solvent. A carrier solvent does not exhibit any absorbance at either the measuring or reference wavelength. This type of detector has a single flow cell (also referred to herein as a sample cell) where the sample in the flow stream is subjected to radiation at both wavelengths. An apparent advantage in using this type of detector is that factors which affect the intensity of one wavelength, other than absorption of optical energy by the sample, would similarly affect the other wavelength. However, in spite of this advantage, typical single beam, dual-wavelength configurations for absorption detection in a continuous-flow sampling system may have inherent limitations which affect its ability to detect absorption efficiently and accurately. Some of these limitations may be found in the typical continuous flow cell, the source of optical energy and the requirement that electrical components and flow components of the absorption detector be placed in close proximity to each other.
Prior art FIGS. 2A and 2B illustrate simplified arrangements used in typical single-beam, dual-wavelength absorption detector for irradiating a sample material (species) in a continuous-flow detector-cell and for detecting the illumination which has been subjected to the sample material. The term "detector-cell" is used herein to avoid confusion with another term "detector". The term "detector-cell" in this specification includes the sample cell and the components associated with it, e.g., a means for source radiation to enter the cell to irradiate the sample, a means for illumination that was subjected to the sample material to exit the sample cell for detection, and a means for the sample material (species) to enter the sample cell; whereas, the term "detector" includes the detector-cell and any other components, including electronics, necessary to produce a signal which is representative of the absorption of the sampled material.
FIG. 2A is a simplified representation of a detector-cell having a continuous-flow sample cell 10a located between a source of optical energy 12 and a photodetector (photodiode or phototransducer) 14. In this arrangement, optical energy is transmitted inline with the sample flow. The length of the optical energy path within the sample cell determines the sensitivity of this absorption detector. The longer the optical path is within the sample cell, the greater the detector's sensitivity to differences in the absorption of optical energy between the measuring wavelength and the reference wavelength.
In FIG. 2A, sample cell 10a is shown in a vertical-longitudinal cross-section. This particular sample cell 10a is known as a "Z" flow-through channel structure. Other channel geometries are possible, for example, FIG. 2B illustrates in vertical-longitudinal cross-section another widely used flow-through sample cell 10b which has a "U" structure. The directions of flow in sample cells 10a and 10b are indicated by the arrows. The optical energy enters the sample cell in each of the FIGs. at window 16 and exits by window 18.
These flow cells (10a and 10b), shown in FIGS. 2A and 2B, are commonly used, but in order for optical radiation to be transmitted inline with the flow path of the sample material, the sample has to follow a tortuous path of flow into and out of the sample cell. One frequent problem with detectors having these types of sample cells is that they are subject to bubble noise. Often the species may have entrained gas bubbles or air bubbles. Bubble noise is caused by the bubbles becoming trapped within the sample cell due to the tortuous path of the sample through the flow cell. The natural buoyancy of the bubble in the fluid may cause it to contact and to adhere to a wall of the flow cell. When the pumping system does not provide enough flow to overcome the adherence and the friction between the bubble and the wall, it may be difficult to dislodge the bubble from the flow cell. The pumping system then causes the bubble to pulsate between pumping cycles, thus causing pulsations in the illumination intensity at photodetector 14.
One solution to the bubble problem is to use another arrangement for the sample cell as shown in FIG. 2C. This sample cell 10c is a straight flow-through cell aligned vertically such that the optical energy is transmitted across the cell, i.e., transverse to the flow. However, in order to maximize sensitivity, the cell is broadened to increase the optical path length. By increasing the optical path length, the volume of the cell is increased. This increases the dead zone of the sample cell. A larger dead zone requires that the individual samples have larger volumes for adequate separation from other samples and to prevent the sample from mixing in the sample cell. This results in reducing the total number of samples that may be done in a given time period and also results in broader absorption peaks, as depicted in absorption peak 1 of FIG. 1, because the sample remains in the sample cell longer.
If the optical path length is decreased to reduce the volume of the cell, the sensitivity of the detector is reduced. For example, assuming everything else remains the same, if the optical path length is halved, then the amount of absorption of optical energy is substantially reduced in both wavelengths, thereby reducing the ratio between the reference and measuring wavelength. Consequently, the output signal (absorption measurement) may be reduced by as much as a half. This is illustrated on FIG. 1, where in this case, the amplitude of the output signal would be represented on a recorder as peak 3, instead of peak 4. Sensitivity is particularly important when a heavily diluted sample is used which does not readily absorb at the measuring wavelength. For example, peaks 6, 7, and 8 on FIG. 1 represent a dual-wavelength sample measurement which has relatively low absorption measurement. If the optical path length were to now be decreased by one half, the peaks may be indistinguishable from noise if the sample line was subject to bubble noise or other forms of noise present in a plant environment.
There are other limitations with the arrangements shown in FIG. 2A, 2B and 2C. Because the light source 12 and photodetector 14 are on opposite sides of the flow cell 10, these arrangements may not effectively utilize the light (radiation) produced by the source, i.e., they do not exhibit high coupling efficiency for collecting light. Light upon entry into the flow cell through window 16 will fan-out (diffuse). Diffusion of light may result from numerous causes. Primarily, in this case, it results from the source producing a beam of light having rays which originate from a plurality of point sources. And, all of these point sources are not aligned so that when the rays of each point source enter the inlet window (window 16), the rays are not exactly aligned with the length of the flow cell and the outlet window 18. Additionally, other forms of diffusion also occur; these forms include: (1) refraction of light as it crosses material boundaries, (2) scattering of light due to particles along the path length and (3) the tendency of light to spread out normal to its path of movement (Beam spreading).
If the flow cell is narrow, as shown in FIGS. 2A and 2B, some light, due to the fan-out (diffusion), may be absorbed in the walls or blocked by the walls of the flow cell. If the flow cell is wide, as shown in FIG. 2C, only a limited portion of the light upon entry into the inlet window 16 will be directed at window 18. If large amounts of optical energy are lost within the flow cell, then the detector will have a reduced ability to detect weak, e.g., strongly absorbed, illumination at the detector. The weakest signal that can be detected by the detector is limited by the "dark" current (noise) produced by the photodetector. As the illumination becomes weaker, the signal to noise ratio is reduced until a point is reached where it is not possible to distinguish between the signal produced by the weak illumination and the noise.
The signal to noise ratio between the detected illumination and the dark current may be increased by increasing the magnitude of the optical energy entering the sample cell. Increasing irradiating illumination increases the maximum repeatable ratio of reference illumination to measuring illumination that is detectable above the noise. However, increasing the amount of light transmitted into the flow cell could also result in increasing the temperature of the sample. Sample heating could cause chemical reactions or bubbles to come out of solution resulting in the sample no longer being representative of the material sampled.
The windows 16, 18 of FIGS. 2A, 2B, and 2C are also sources of inherent design limitations. They may be an integral part of the flow cell or they may be removable. In any case, they become dirty or scarred from the sample material. This reduces the performance of the absorption detector. The flow cell or the separately attached windows must be cleaned or replaced. Whether the windows are an integral part of the flow cell or separately attached, the windows are difficult to inspect, clean or replace in typical detector-cell arrangements.
Safety concerns related to electrical components of the detector-cell and ageing of the optical energy source also affect the utility of this type of detector. The cost to install this absorbance detection system could be expensive due to the expenditures necessary to meet fire and building codes. These safety codes are necessary because: (1) the sample cell may contain hazardous and/or explosive materials when sampling, and/or (2) the detector's electrical circuitry, which powers the optical energy source and the photodetector, could ignite hazardous or explosive vapors in the local area or vapors from the sample cell. When the arrangements shown in FIGS. 2A, 2B and 2C are used for in-plant monitoring, the radiation source, the photodetector and the detector-cell are housed in separate compartments with the compartments being located adjacently to, and inline with, each other so that optical energy may be transmitted between the compartments. The need to locate these components near each other and within the line of sight of each other, yet separate them to meet safety codes, substantially reduces the options available in locating an absorption detector within a processing environment.
In addition, many dual-wavelength absorption detectors use an optical energy source which has a wide frequency band. Special optical components such as optical filters and beam splitters are necessary to separate the reference and sampling wavelengths to determine the absorption thereby increasing the cost of the instrument. Also, as the wide band frequency source ages, the relative power levels of the frequencies within its spectrum change. This is called frequency spectrum shift and results over time in reducing the validity of dual-wavelength absorption measurements.
A simple, compact, robust and inexpensive dual-wavelength absorption detector is needed which features a configuration that (1) reduces bubble noise, (2) compensates for and utilizes the diffusion of source illumination, (3) allows easy access to the windows for inspection or replacement, (4) increases the sensitivity of the detector without increasing the dead zone of the sample cell and (5) provides for greater selectivity in locating absorption detector components to meet safety codes.